The interferometric Photo-Activated Localization Microscopy (iPALM) technique can be regarded as a refinement of the conventional (non-interferometric) PALM technique, whereby the former augments the latter with the ability to perform resolution/image reconstruction axially as well as laterally. The augmentation can be understood in several ways, as discussed below.
In PALM, lateral super-resolution is achieved by sequentially exciting spatially sparse subsets of objects (photoactivatable fluorophores) in a specimen, causing temporal separation of fluorescence emission from these different subsets. The resolvability of objects within each of these sparse subsets is greater than if the whole specimen were to be imaged in one go. In essence, the resolution-limiting diffraction effects that one would expect if one were to attempt to simultaneously image a dense set of objects are circumvented by instead regarding the set as a cumulative collective of sparse subsets, which are sequentially imaged. The photoactivatable fluorophores are caused to fluoresce in a two-step process, whereby in a preliminary step, a so-called “activating wavelength” (or “activation wavelength”) is used to promote the fluorophore from a non-emissive to an emissive state; and in a subsequent step, a so-called “exciting wavelength” (or “excitation wavelength”) is used to cause radiative “relaxation” of the activated fluorophore (fluorescence excitation).
In iPALM, the lateral (XY) super-resolution achieved in PALM is taken a step further, by introducing a mechanism that will also allow fine axial/depth (Z) resolution. This is achieved by imaging the (fluorophores in the) specimen through a pair of oppositely disposed projection systems (objective lenses, optical columns), whose output beams are fed into an optical combining element (specifically, a three-phase beam splitter), where they optically interfere. The resulting interference fringe pattern will be (very) sensitive to the axial (depth) position of the object (fluorophore) being imaged, since this will influence the relative path lengths of the interfering beams. By using a detector arrangement comprising multiple detectors (e.g. CCDs) to selectively look at phase-separated outputs from the combining element, one can effectively (mathematically) “translate” a given fringe pattern into a deduced axial object position; in iPALM, three distinct outputs from the combining element (mutually phase-shifted by 120°) are observed using three different detectors (cameras), whereby the relative intensities of the outputs observed by these cameras will change in a predictable manner as a function of axial fluorophore position.
Although iPALM is a useful technique, it does suffer from drawbacks. More specifically, it relies on a relatively complicated optical/detection architecture. In particular the employed three-phase beam splitter is an expensive and fragile component that is difficult to manufacture. Its performance is sensitive to temperature fluctuations and mechanical vibrations, and it has a relatively long settling time after being disturbed. Moreover, it is difficult to optically align/adjust. The employed three-phase beam splitter is also difficult to mechanically scale up in size, e.g. to match cameras with a larger field of view (without vignetting). Limiting factors in this regard include tolerances on the planar optics of the beam splitter, and coherence characteristics of the fluorescence light. The detection set-up requires the use of three detectors/cameras, which increases bulk/decreases available space, and increases expense.
One should note the distinction between a wide-field microscope—which can be regarded as employing a planar imaging wave—and, for example, a point scanning microscope (German: “Rastermikroscop”), which uses an imaging beam that is focused to a point, and is thus (necessarily) scanned over an object to be imaged. The present invention relates to the former (wide field).
It is an object of the invention to address these issues. In particular, it is an object of the invention to provide an alternative depth-resolved localization microscopy technique that utilizes a radically different illumination/detection configuration. More specifically, it is an object of the invention that does not require use of a three-phase beam splitter.
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